ARTICLE OPEN ACCESS Neopterin is associated with hippocampal subfield volumes and cognition in HIV

Debra A. Fleischman, PhD, Konstantinos Arfanakis, PhD, Sue Leurgans, PhD, Sheila M. Keating, PhD, Correspondence Melissa Lamar, PhD, David A. Bennett, MD, Oluwatoyin M. Adeyemi, MD, and Lisa L. Barnes, PhD Dr. Fleischman [email protected] Neurol Neuroimmunol Neuroinflamm 2018;5:e467. doi:10.1212/NXI.0000000000000467 Abstract Objective HIV infection sets off an immediate immune response and inflammatory cascade that can lead to neuronal injury and cognitive impairment, but the relationship between immune markers, regional brain volumes, and cognition remains understudied in HIV-infected adults.

Methods Cross-sectional associations were examined between serum immune markers of activation (neopterin) and inflammation (interleukin [IL]-1β, IL-6, tumor necrosis factor alpha, and C-reactive protein) with regional brain volumes (cortical, subcortical, total gray matter, hippocampus, and subfields) and cognition in 66 HIV-infected, virally suppressed, adults who underwent 3.0-T MRI as part of the Research Core of the Rush Center of Excellence on Disparities in HIV and Aging. Immune markers were assayed from frozen plasma, values were entered into linear regression models as predictors of regional brain volumes, and interactive effects of immune response and regional brain volumes on cognition were examined.

Results No inflammatory marker was associated with any regional brain volume. Higher neopterin level was associated with lower total hippocampal, presubiculum, and cornu ammonis (CA) subfield volumes. Higher neopterin level and lower total hippocampal volume were independently associated with lower episodic memory, and neopterin level fully mediated the effect of hippocampal atrophy on episodic memory. Higher neopterin levels were associated with lower presubiculum, CA1, and CA4/dentate volumes and lower semantic memory, working memory, and global cognition.

Conclusion Immune activation in response to HIV infection, measured by neopterin, has a deleterious and targeted effect on regional brain structure, which can be visualized with clinically available MRI measures of hippocampus and its subfields, and this effect is associated with lower cognitive function.

From the Rush Alzheimer’s Disease Center (D.A.F., K.A., S.L., M.L., D.A.B., L.L.B.), Rush University Medical Center; the Department of Neurological Sciences (D.A.F., S.L., M.L., D.A.B., L.L.B.), the Department of Behavioral Sciences (D.A.F., M.L., L.L.B.), the Department of Preventive Medicine (S.L.), the Department of Diagnostic Radiology and Nuclear Medicine (K.A.), Rush University Medical Center; Ruth M. Rothstein CORE Center (O.M.A.); the Biomedical Engineering (K.A.), Illinois Institute of Technology, Chicago; the Blood Systems Research Institute (S.M.K.), San Francisco, CA; and the University of California at San Francisco (S.M.K.), Laboratory Medicine.

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Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CA = cornu ammonis; CEDHA = Center of Excellence on Disparities in HIV and Aging; CRP = C-reactive protein; CV = coefficient of variation; FDR = false discovery rate; HAART = highly active antiretroviral therapy; IL = interleukin; INF = interferon; TNFα = tumor necrosis factor alpha.

It has long been known that HIV infection produces an almost Methods immediate immune activation and inflammatory cascade that can persist in the face of effective highly active antiretroviral Participants therapy (HAART) treatment1 and can lead to neuronal in- The study was conducted among participants in the Research jury2 and cognitive impairment.3 The earliest phase of the Core of the Rush Center of Excellence on Disparities in HIV HIV immune response occurs when interferon-stimulated and Aging (CEDHA), a longitudinal study of older adults (IFN-γ) T-lymphocytes activate monocytes, macrophages, with HIV infection. HIV+ participants (n = 175) were recruited from an urban infectious disease clinic. Eligibility and dendritic cells to produce pteridines, a class of chemicals fl requirements included documented HIV infection, African that stimulate the in ammatory cascade through the release of + ≥ 3 β American, or white, age 50 and older, CD4 200 cells/mm cytokines (e.g., interleukin [IL]-1 , IL-6, and tumor necrosis + ≥ α on HAART or CD4 500 HAART naive, and viral loads factor alpha [TNF ]) and acute phase reactants (e.g., ranging from undetectable up to 50,000 copies. All partic- C-reactive protein [CRP]). ipants underwent annual structured clinical evaluations that included an assessment of risk factors for cognitive decline, Neopterin, a pteridine produced in monocyte-derived mac- a battery of cognitive function tests, and a blood draw. rophages4 and dendritic cells,5 is a well-established early 6 marker of HIV-associated cellular immune activation. The HIV+ participants who were willing to undergo MRI were neopterin level correlates with measures of viral load and approached if they were nondemented and able to give in- ffi CD4 count, is a sensitive measure of treatment e cacy and formed consent. Participants were excluded if there was any 7–9 10 HIV disease course, and predicts HIV-related mortality. contraindication for MRI, history of head injury with loss of Neopterin level is associated with compromise of blood- consciousness over 30 minutes, known large lesions, MS or brain barrier integrity and generalized brain atrophy in HIV11 hydrocephalus, seizure disorder, schizophrenia, current use of and HIV-associated cognitive impairment.2 Thus, neopterin neuroleptic, narcotic medication, or anti-inflammatory med- level may signal activation of HIV-related immune mecha- ications. Seventy-eight of the 175 participants recruited into nisms that can lead to neuronal injury, perhaps in regions the research core were ineligible based on the above criteria. most susceptible to metabolic disturbance, neurologic dis- Of those 78 remaining, 11 participants were eligible but could ease and alterations in cognitive function, but no study of the not be contacted, 11 participants were reluctant to scan, 5 had finer associations of the neopterin level with MRI-derived unsuccessful scans, and 4 did not have blood available for fi regional brain volumes and cognition in HIV has been assay, making the nal sample 66 participants. reported. Standard protocol approvals, registrations, Here, we examined relationships between immune activa- and patient consents tion measured by serum neopterin, inflammation measured The study was approved by the Rush Institutional Review by serum markers (IL-1β,IL-6,TNF-α,andCRP)known Board, and all participants signed study consent. to be present in the HIV immune response, even with Cognitive testing successful HAART treatment, and associated with brain – Each participant underwent cognitive function testing that abnormalities and cognitive impairment and decline,12 143T- included 18 tests of episodic memory, semantic memory, MRI-derived regional brain structure, and cognition in HIV- working memory, perceptual speed, and visuospatial ability.17 infected adults. Given the evidence that HIV is associated See supplementary materials for details, links.lww.com/ with increased levels of immune activation and chronic in- NXI/A47. flammation, we tested the hypothesis that higher level of immune activation, measured by neopterin, and higher in- Serum immune activation and fl fl ammation, measured by these 4 in ammatory markers, inflammatory markers fi would be speci cally associated with volume loss in the Neopterin was measured in undiluted plasma by ELISA hippocampus, a brain region known to be highly susceptible (Alpco, Salem, NH; standard curve measuring 0.5–63 ng/mL 15 to metabolic stress and neurologic disease and HIV in- with 3% intraplate and 7% interplate coefficient of variation fection.16 Furthermore, we hypothesized that this volume [CV]%). ELISA results were acquired using Molecular reduction would be associated with lower episodic memory, Devices E-Max plate reader with Softmax Pro v5.4. MILLI- which depends critically on hippocampal integrity. PLEX MAP Human High Sensitivity T Cell Panel was used to

2 Neurology: Neuroimmunology & Neuroinflammation | Volume 5, Number 4 | July 2018 Neurology.org/NN measure IL-1β, IL-6, and TNF-α in undiluted plasma (Milli- First, we examined correlations between the demographic, pore, Billerica, MA; standard curve measuring 0.18–2,000 pg/ disease, and immune marker variables No immune marker or mL with 7% intra- and inter-plate CV). MILLIPLEX MAP disease variable was associated with race or age (ps > 0.05), Cardiovascular Disease Panel 3 was used to measure CRP in and females had higher CRP level (female: mean [log10] 4.17, diluted plasma (1: 40,000) (standard curve measuring SD = 0.55; male: mean = 3.81, SD = 0.52, t = 2.33, p = 0.023). 0.01–50 ng/mL with 1% intraplate CV% and 8% interplate CV%). See supplementary materials for details, link- Persons with higher neopterin level had lower CD4 counts s.lww.com/NXI/A47. (r = −0.369, p < 0.01), but there was no association with nadir CD4 or disease duration. No inflammatory marker was as- Brain image acquisition and processing sociated with CD4 or any other disease variable. The in- All MRI data were collected on a 3-T Philips MRI scanner flammatory markers were significantly interrelated, but none (Best, Netherlands) with a 32-channel head coil. See supple- was related to neopterin. Correlations between the immune mentary materials for further details (links.lww.com/NXI/A47) marker variables are shown in supplementary materials, table 18 on MRI acquisition and postprocessing. e-1, links.lww.com/NXI/A47.

Statistical approach Next, we examined correlations between the demographic MRI data were transferred to SAS (SAS Institute, Inc., Cary, and disease severity variables with regional brain volumes. All NC, 2012) for further data manipulation, linkage with de- selected regions had higher volumes with lower age and mographic and serum immune marker data, and statistical higher CD4 count (ps < 0.05). There were no significant ffi analyses. Mean, SD, skewness coe cients, Q-Q plots, and associations with sex or race. histograms were obtained for age, education, CD4, serum markers, brain, and cognitive measures. Because the dis- In a set of linear regression models, corrected for de- tributions of neopterin and CRP were markedly skewed, mographics (age, education, sex, and race) and CD4, we next logarithmic transformations (base 10) were used in all anal- examined associations between immune markers and total yses. Because the other 3 markers were below the detection cortical, total subcortical, total gray, and total hippocampal limit in many participants, we used Spearman correlations in volume. No immune marker was associated with total cor- descriptive statistics, and we used dichotomized values tical, total subcortical, or total gray matter volume. Persons (detected/not detected) in analytic models. First, we exam- with higher neopterin levels had lower total hippocampal ined the correlations between demographic and disease volumes (estimate = 0.398, SE = 0.128, p = 0.003). Estimates, characteristics with immune markers and with brain variables; SEs, and p values are shown in table 2. The full model for then, we used regression models (with brain measures as neopterin and total hippocampal volume is shown in sup- outcomes) to examine the association of each of the immune plementary materials, table e-2, links.lww.com/NXI/A47. markers with whole-brain measures (total cortical, total sub- Because neopterin was the only immune marker associated cortical, total gray, and total hippocampus) with terms to with brain volume, no further analyses were performed with correct for age, education, race, sex, and CD4 level. Based on inflammatory markers. The remaining analyses concentrated fi the ndings, we then examined the associations of neopterin on associations between neopterin, regional hippocampal fi with hippocampal sub eld volumes, with false discovery rate volumes, and cognition. (FDR) corrections for multiple comparisons. Finally, because of known associations of hippocampus volume with memory, Next, a finer examination of the associations of neopterin with fi we rst considered episodic memory as the outcome of hippocampal volume was undertaken using 7 subfield vol- a multiple regression model with terms for neopterin and umes: presubiculum, subiculum, cornu ammonis (CA)1, fi sub eld volume and their interaction. In secondary models, CA2/3, CA4/dentate, fimbria, and hippocampal fissure. we replaced episodic memory with semantic memory, work- Volumes of presubiculum, subiculum, and fimbria were higher ing memory, perceptual speed, and visuospatial ability. All with lower age (ps < 0.05). There were no associations with ffi regression coe cients are reported per SD of the outcome; sex or race. Persons with higher CD4 count had higher vol- ffi for continuous covariates, the regression coe cients are based ume in all fields except the hippocampal fissure (ps < 0.05). on standardized covariates. In a series of linear regression models, corrected for de- Data availability mographic variables and CD4, persons with higher neopterin Anonymized data not published within this article will be level had lower volumes of presubiculum, and the CA (CA1, shared by request from any qualified investigator. CA2/3, and CA4/dentate) fields, after correcting probability levels for multiple comparisons using FDR. Estimates, SEs, Results and p values for the regression models are shown in table 3. Descriptive information for demographic, disease status, im- As a final step, we examined the associations of neopterin and mune markers, brain volume, and cognitive measures is given regional brain volumes first with episodic memory in separate in table 1. linear regression models controlled for demographic and

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 5, Number 4 | July 2018 3 Table 1 Demographic characteristics, HIV disease status, immune, brain, and cognitive measures (n = 66)

Measure Mean SD Minimum Maximum

Age at scan 58.5 5.3 50.6 71.3

Years education 13.5 3.2 8.0 24.0

Male, n (%) 48 (73%)

Black, n (%) 45 (68%)

CD4 638.5 313.5 228.0 1,872.0

Nadir CD4a 276.4 191.2 6.0 756.0

Disease duration (y) 16.0 7.1 1.0 32.0

Neopterinb 0.42 0.17 0.14 0.88

IL-1β, n (%) 35 (53%)

IL-6, n (%) 44 (67%)

TNF-α, n (%) 52 (79%)

CRPc 3.91 0.58 2.65 5.09

Cortical gray volumed 262.99 21.58 211.03 308.28

Subcortical gray volume 36.09 3.82 28.60 44.64

Total gray volume 358.38 30.61 287.20 414.26

Hippocampal volume 5.14 0.71 3.61 6.87

Presubiculum 0.60 0.09 0.42 0.81

Subiculum 0.80 0.11 0.58 1.10

CA1 0.42 0.05 0.29 0.53

CA2/3 1.74 0.17 0.87 1.63

CA4/dentate 0.70 0.10 0.50 0.92

Fimbria 0.07 0.03 0.01 0.12

Hippocampal fissure 0.06 0.03 0.02 0.16

Global cognitionc 0.15 0.55 −0.97 1.22

Episodic memory 0.25 0.67 −1.26 1.59

Semantic memory 0.07 0.79 −1.65 1.56

Working memory 0.16 0.78 −1.43 1.85

Perceptual speed 0.09 0.82 −1.68 1.85

Visuospatial ability 0.04 0.80 −1.38 1.57

Abbreviations: CA = cornu ammonis; CRP = C-reactive protein; IL = interleukin; TNF-α = tumor necrosis factor alpha. a n = 53. b Log10-transform of units. c All brain volumes are tenths of percents of intracranial volume. d All cognitive scores converted to Z-scores (mean = 0).

disease variables, and then, in secondary analyses, with global attenuated, but not eliminated (estimate = −0.284, SE = cognition, semantic memory, working memory, perceptual 0.135, p = 0.039), but the association between hippocampal speed, and visuospatial ability. Persons with lower neopterin volume and episodic memory was eliminated (p = 0.22), levels (estimate = −0.347, SE = 0.126, p < 0.01) and higher suggesting that neopterin level fully mediated the association hippocampal volumes (estimate = 0.257, SE = 0.121, p < 0.05) between hippocampal volume and episodic memory. The had better episodic memory. When neopterin and hippo- interaction of neopterin and hippocampal volume was not campal volumes were entered in a single model, the associ- significant. Full models are shown in supplementary mate- ation between neopterin and episodic memory was slightly rials, table e-3, links.lww.com/NXI/A47.

4 Neurology: Neuroimmunology & Neuroinflammation | Volume 5, Number 4 | July 2018 Neurology.org/NN Table 2 Associations of immune markers and brain volumes (estimate, SE)a

Cortical gray Subcortical gray Total gray Hippocampus

Neopterin −0.223 (0.127) −0.080 (0.131) −0.172 (0.130) −0.398 (0.128)b

IL-1β 0.049 (0.236) 0.230 (0.236) 0.093 (0.239) 0.072 (0.252)

IL-6 0.142 (0.241) 0.240 (0.241) 0.195 (0.244) 0.101 (0.256)

TNF-α −0.111 (0.279) −0.049 (0.281) −0.089 (0.284) −0.156 (0.297)

CRP −0.073 (0.116) 0.051 (0.117) −0.041 (0.118) 0.022 (0.123)

Abbreviations: CRP = C-reactive protein; IL = interleukin; TNF-α = tumor necrosis factor alpha. a Regression coefficients from separate models for brain volumes with a term for an immune marker, models controlled for age, sex, education, race, and CD4 status. IL1-β, IL-6, and TNF-α were included as binary variables (detected/not detected), and the coefficient is scaled to the SD of the brain volume. For neopterin and CRP, the coefficients are standardized regression coefficients. b p < 0.01.

Interactions between the neopterin level and hippocampal lower total hippocampal volume and lower presubiculum and subfield volumes were statistically significant for other cognitive CA subfield volumes. Episodic memory was better for persons domains. The standardized interaction term of the neopterin with lower neopterin levels and for persons with higher hip- level with CA1 for semantic memory (estimate = 0.321, SE = pocampal volumes. In mediation analyses, neopterin level 0.114, p < 0.01), CA4/dentate for semantic memory (estimate fully accounted for the effect of total hippocampal volume on = 0.29, SE = 0.14, p = 0.04), presubiculum for global cognition episodic memory. In hippocampal subfield analyses, persons (estimate = 0.215, SE = 0.106, p = 0.047), and presubiculum for with higher neopterin levels had lower CA1, CA4/dentate, working memory (estimate = 0.262, SE = 0.121, p =0.034), and presubiculum volumes and lower semantic memory, were all statistically significant. Figure 1 illustrates the associa- working memory, and global cognition. tion of hippocampal subfield volumes to cognition for the 2 extreme quintiles (10th and 90th) of the neopterin level. The Neopterin production is the complex result of direct stimu- observation that the slopes for low levels of neopterin are lation from IFN-γ, the only cytokine that directly induces it,19 negative and the slopes for high levels of neopterin are positive and numerous indirect effects and interactions between reflects the fact that all the interaction terms are positive. multiple cytokines and thus reflects the entirety of immune network response on monocytes, macrophages, and dendritic cells.20Neopterin is often elevated before antibody serocon- Discussion version or the onset of any clinical symptoms21 and hence may be an earlier and more sensitive indicator of HIV- In this study, 66 HIV-infected adults who had higher serum associated immune response than any single, or any set of, neopterin levels, a measure of cellular immune activation, had 6,20 inflammatory markers. In this study, we measured 4 in- flammatory markers that are known to be present in HIV infection and associated with brain abnormalities and cogni- Table 3 Associations of neopterin and hippocampal tive impairment and decline, IL-1β, IL-6, TNF-α, and CRP, a subfield volumes (estimate, SE) but none was associated with brain volume. Only neopterin, Subfield Estimate SE Corrected p valueb a marker for immune activation, was associated with CD4, a marker of disease severity, and with regional brain volumes, − Presubiculum 0.31 0.12 0.015 specifically the hippocampus and its subfields. Subiculum −0.30 0.13 0.061

CA1 −0.46 0.14 0.002 No brain region is as susceptible to noxious insult as the hippocampus.15 The pyramidal neurons of the CA subfields, CA2/3 −0.46 0.14 0.002 particularly CA1,22 are well known to be vulnerable to 23 24 CA4/dentate −0.46 0.13 0.003 anoxia-ischemia, metabolic stress (e.g., hypoglycemia ), microvascular pathology,25 and neurodegenerative disease Fimbria −0.19 0.11 0.135 such as Alzheimer disease and vascular dementia.26 HIV viral Fissure −0.18 1.28 1.089 load is high in the hippocampus27 and its subfields, with pyramidal neurons in CA3 possibly being more vulnerable to Abbreviation: CA = cornuammonis. 16 a Estimated from separate models of standardized hippocampal subfield the virus than CA1, and reduced hippocampal volumes outcome with a term for standardized neopterin, controlled for age, sex, 28 education, race, and CD4 status. Thus, all coefficients in this table are have been demonstrated in HIV-infected adults. The expressed as standardized regression coefficients. results of this study suggest that hippocampal atrophy that b FDR (false discovery rate) corrected for multiple comparisons. Bold values are statistically significant. occurs in HIV is associated with immune activation, as measured by neopterin.

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 5, Number 4 | July 2018 5 Figure Association of hippocampal subfield volumes to cognition by neopterin level, volumes, and cognition adjusted for age, education, race, sex, and CD4

Colored lines show estimated cognition vs volume for extreme percentiles of neopterin (90th percentile in blue and 10th percentile in red). Solid points are data for persons in extreme quintiles (blue, 5th quintile; red, 1st quintile). Data for persons in the middle quintiles of neopterin are shown in open circles.

Hippocampal integrity is critical for normal memory func- of that information.31 The results of this study suggest that tion,29 and in this study, hippocampal volume was, indeed, immune activation in HIV-infected adults, measured by positively associated with episodic memory. Persons with neopterin, may signal early volume reductions in hippocampal higher neopterin levels had worse episodic memory, and, in subfields that are responsible for normal encoding and re- mediation analyses, the neopterin level fully accounted for the trieval of declarative information. effect of hippocampal atrophy on episodic memory. These results suggest that immune activation, as measured by neo- Although it has long been known that the hippocampus is pterin, has a clear and robust deleterious effect on episodic critical for the encoding and retrieval of long-term declarative memory in HIV-infected adults. memories, more recently, the region has been shown to play a central role in other cognitive processes such as working In hippocampal subfield analyses, persons with higher neo- memory32 or the ability to maintain and manipulate multiple pterin levels had lower volumes in 2 CA subfields (CA1 and items simultaneously in short-term memory. The hippo- CA/4dentate) and lower semantic memory. Episodic and campus is recruited in complex working memory span tasks semantic memory are considered long-term, declarative that involve processing relations between item features,33 and memory processes known to be strongly dependent on hip- the subiculum subfield has been shown to be preferentially pocampal circuitry,30 particularly the CA subfields, with CA4/ activated in response to discriminating highly associated fea- dentate known to be a critical region for encoding in- tures from multiple viewpoints in a scene discrimination formation, and CA1 known to be a critical region for retrieval task.34 In this study, persons with higher levels of neopterin

6 Neurology: Neuroimmunology & Neuroinflammation | Volume 5, Number 4 | July 2018 Neurology.org/NN had lower volumes of the presubiculum, 1 of 4 component markers were highly intercorrelated, and not related to neo- regions of the subiculum, and lower working memory and pterin, and associations with neopterin were robust, sug- global cognition. This finding is consistent with the notion gesting that the neopterin level may, indeed, be a window on that immune activation, measured by neopterin, may flag an upstream immune response that is potentially damaging to HIV-associated neuronal injury in a vulnerable subfield and the integrity of brain volume and cognition. Third, we used consequent risk for global cognition and working memory serum neopterin rather than CSF neopterin; however, impairments. Overall, the results of this study suggest that a number of studies have shown that serum and CSF neo- neopterin level may serve as a proxy for ongoing risk of HIV- pterin are correlated21 and that serum neopterin parallels HIV associated brain injury and neurocognitive disorder, but lon- RNA levels.40 Fourth, the age range of our sample was limited, gitudinal studies are needed. and the findings cannot be generalized to much younger or older HIV-infected adults. Fifth, the analyses did not adjust There are multiple mechanisms potentially linking neopterin for premorbid general intelligence, which could influence biology with neuronal damage, neurodegeneration, and con- individual level of cognitive reserve available to participants sequent cognitive impairment in HIV infection. One candi- after contracting HIV. Sixth, the study is cross-sectional and date mechanism is the disruption by chronic immune therefore cannot demonstrate a causative relationship be- activation of the kynurenine pathway of tryptophan oxidative tween neopterin level and brain volume and cognition. Fi- metabolism, which can induce cell damage through dysregu- nally, although our sample is small and cross-sectional, it was lation of NMDA receptor function or generation of reactive exceptionally well characterized within the parent CEDHA oxygen species.35 Higher neopterin level is associated with study, which is a longitudinal prospective study of HIV, and lower serum tryptophan, brain atrophy, and dementia in HIV we will be able to perform longitudinal analyses in the future. infection,11 and neurotoxic glutamate is known to be elevated Limitations notwithstanding, the results of this study suggest in the CSF and plasma of patients with HIV.36CA1 hippo- that the serum neopterin level and 3T neuroimaging, both of campal cells have a high expression of NMDA receptors which are clinically available and relatively easy to obtain, may (which are critical for learning and memory) and are thus be useful in identifying those HIV-infected adults who are at vulnerable to glutamate-mediated excitotoxicity.37 Indeed, the risk of developing further neuronal injury and cognitive im- integrity of this pathway has been implicated in other medical pairment. This information could prove invaluable for early conditions that involve chronic immune activation, brain in- implementation of targeted pharmacologic and behavioral jury, and cognitive impairment including Alzheimer disease,38 interventions. metabolic syndrome, and vascular cognitive impairment.39 Thus, it is feasible that chronic immune activation in HIV Author contributions infection could be leading to neuronal loss and diminished Study concept and design: D.A. Fleischman and K. Arfanakis. cognitive function through an effect on the kynurenine Analysis or interpretation of the data: D.A. Fleischman, pathway, but the biological mechanisms underlying the as- K.Arfanakis,S.Leurgans,S.M.Keating,M.Lamar,D.A. sociation between chronic immune activation, brain integrity, Bennett, and L.L. Barnes. Drafting of the manuscript: D.A. and cognitive function in HIV infection are complex and re- Fleischman,K.Arfanakis,S.Leurgans,S.M.Keating, main understudied. M. Lamar, D.A. Bennett, and L.L. Barnes. Statistical analysis: S. Leurgans. Obtained funding: D.A. Fleischman, K. Arfanakis, This study had important strengths and limitations. First, the D.A. Bennett, O.M. Adeyemi, and L.L. Barnes. immune markers for this study were selected to target im- mune activation and inflammation, but only neopterin was Acknowledgment associated with regional brain neuronal injury and cognitive The authors thank the staff of the Rush Center of Excellence impairment. Considering that the study participants were fully on Disparities in HIV and Aging, the research staff of the Ruth virally suppressed, low levels of plasma inflammatory markers M. Rothstein Core Center, and the staff of the Rush may not have the dynamic range to identify associations with Alzheimer’s Center, particularly Woojeong Bang, MS, for markers of HIV-induced pathology. Low concentrations for statistical analysis and Niranjini Rajendran, MS, for image these cytokines are not uncommon in well-controlled HIV postprocessing. infection, and it is known that there is variable detectability of IL-6, TNF-α, and IL-1β in HIV-infected individuals. The ro- Study funding bust associations of neopterin with regional brain volumes This study was supported by the National Institute of Mi- and cognition shown here support previous studies demon- nority Health and Health Disparities grant P20MD6886; strating that subclinical CNS damage and consequent cogni- National Institute of Aging grant P30AG010161; and Illinois tive impairment do occur in HIV even when the virus is well Department of Public Health. controlled.2 Second, the number of subjects was small, and the lack of associations between regional brain volumes and Disclosure the chosen inflammatory markers may be due to inadequate D.A. Fleischman received research support from the National power, especially for the effects of the interaction of neopterin Institute of Minority Health and Health Disparities. K. Arfanakis with brain volumes on cognition. However, the inflammatory served on the editorial board of Brain Imaging and Behavior and

Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 5, Number 4 | July 2018 7 received research support from the NIA and NINDS. S. Leur- 14. Cox DE, Hoffman SC, DiMercurio BS, et al. Cytokine polymorphic analyses indicate ethnic differences in the allelic distribution of interleukin-2 and interleukin-6. gans serves as an associate editor of statistics for Neurology and Transplantation 2001;72:720–726. received research support from the NIH. S.M. Keating received 15. Bartsch T. The hippocampus in neurological disease. In: Bartsch T, editor. The Clinical Neurobiology of the Hippocampus: An Integrative View. Oxford, United research support from the NIH/NHLBI, NIH/NIAID, US Kingdom: Oxford University Press; 2012:200–205. FDA, amfAR Institute for HIV Cure. M. Lamar received re- 16. Torres-Munoz J, Stockton P, Tacoronte N, Roberts B, Maronpot RR, Petito CK. search support from the NIA, NINDS, and NHLBI. D.A. 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8 Neurology: Neuroimmunology & Neuroinflammation | Volume 5, Number 4 | July 2018 Neurology.org/NN Neopterin is associated with hippocampal subfield volumes and cognition in HIV Debra A. Fleischman, Konstantinos Arfanakis, Sue Leurgans, et al. Neurol Neuroimmunol Neuroinflamm 2018;5; DOI 10.1212/NXI.0000000000000467

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References This article cites 39 articles, 8 of which you can access for free at: http://nn.neurology.org/content/5/4/e467.full.html##ref-list-1 Subspecialty Collections This article, along with others on similar topics, appears in the following collection(s): All Clinical Neurology http://nn.neurology.org//cgi/collection/all_clinical_neurology All Cognitive Disorders/Dementia http://nn.neurology.org//cgi/collection/all_cognitive_disorders_dementi a All Immunology http://nn.neurology.org//cgi/collection/all_immunology HIV http://nn.neurology.org//cgi/collection/hiv Volumetric MRI http://nn.neurology.org//cgi/collection/volumetric_mri Permissions & Licensing Information about reproducing this article in parts (figures,tables) or in its entirety can be found online at: http://nn.neurology.org/misc/about.xhtml#permissions Reprints Information about ordering reprints can be found online: http://nn.neurology.org/misc/addir.xhtml#reprintsus

Neurol Neuroimmunol Neuroinflamm is an official journal of the American Academy of Neurology. Published since April 2014, it is an open-access, online-only, continuous publication journal. Copyright Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.. All rights reserved. Online ISSN: 2332-7812.